The overall objective of this project is to understand the functional consequences of the unique orbital fibroblast phenotype in the pathogenesis of thyroid-associated ophthalmopathy (TAO), a self-limited aspect of Graves'disease. Central to TAO is anatomic-site selective inflammation and orbital tissue expansion caused by increased adipogenesis and accumulation of hyaluronan. Our central hypothesis is that orbital susceptibility to TAO derives from the attributes of orbital fibroblasts. We have discovered that they are heterogeneous with respect to surface display of the glycoprotein marker, Thy-1. Each subset possesses a distinct capacity to differentiate into either myofibroblasts (Thy-1+) or adipocytes (Thy-1"). Moreover, a disproportionate fraction of TAO orbital fibroblasts express the insulin-like growth factor-1 receptor (IGF-1R). Circulating IGF-1R activating antibody (GD-IgG) can be detected in almost all patients with Graves'disease. GD-lgG/IGF-1R interactions result in the synthesis of powerful T cell chemoattractants, IL-16 and RANTES. Patients with TAO also have more IGF-1R expressing fibroblast precursors (fibrocytes) in their blood. We hypothesize that fibrocytes are recruited to the inflamed orbit in TAO. Orbital fibroblasts exhibit exaggerated responses to IL-1 (3. We hypothesize that these responses result from poor expression of IL- 1 receptor antagonist (IL-1RA). TAO orbital fibroblasts also express 15-lipoxygenase-1 (15-LOX-1)and generate 15-HETE in response to IL-4 or IL-13, Th2 cytokines associated with fibrosis and inflammation dampening. We hypothesize that 15-LOX-1 self-limits inflammation in TAO. Our Specific Aims are 1) characterize the impact of IGF-1 and GD-IgG provoked IGF-1R activation on site-specific adipogenesis and hyaluronan synthesis in Thy-1+ and Thy-1" TAO orbital fibroblasts;2) characterize phenotypes of fibrocytes from patients with TAO by determining their differentiation potential, display of IGF-1 R and responses to GD- IgG;3) determine the molecular basis for exaggerated responses to IL-1p by studying IL-1RA isoform expression, IL-1 receptor display and signaling;4) determine the molecular mechanism and functional implications of anatomic site-selective expression of 15-LOX-1 and the impact of its products, 15-HETE and LXA4 on the actions of IL-1 p. We believe that insights gained from the proposed studies will enhance our understanding of potentially sight-threatening TAO and allow us to develop novel therapeutic approaches.